title: Critical role of anti-apoptotic Bcl-2 protein phosphorylation in mitotic death.
abstract: Microtubule inhibiting agents (MIAs) characteristically induce phosphorylation of the major anti-apoptotic Bcl-2 family members Mcl-1, Bcl-2 and Bcl-xL, and although this leads to Mcl-1 degradation, the role of Bcl-2/Bcl-xL phosphorylation in mitotic death has remained controversial. This is in part due to variation in MIA sensitivity among cancer cell lines, the dependency of cell fate on drug concentration and uncertainty about the modes of cell death occurring, thus making comparisons of published reports difficult. To circumvent problems associated with MIAs, we used siRNA knockdown of the anaphase-promoting complex activator, Cdc20, as a defined molecular system to investigate the role, specifically in mitotic death, of individual anti-apoptotic Bcl-2 proteins and their phosphorylated forms. We show that Cdc20 knockdown in HeLa cells induces mitotic arrest and subsequent mitotic death. Knockdown of Cdc20 in HeLa cells stably overexpressing untagged wild-type Bcl-2, Bcl-xL or Mcl-1 promoted phosphorylation of the overexpressed proteins in parallel with their endogenous counterparts. Overexpression of Bcl-2 or Bcl-xL blocked mitotic death induced by Cdc20 knockdown; phospho-defective mutants were more protective than wild-type proteins, and phospho-mimic Bcl-xL was unable to block mitotic death. Overexpressed Mcl-1 failed to protect from Cdc20 siRNA-mediated death, as the overexpressed protein was susceptible to degradation similar to endogenous Mcl-1. These results provide compelling evidence that phosphorylation of anti-apoptotic Bcl-2 proteins has a critical role in regulation of mitotic death. These findings make an important contribution toward our understanding of the molecular mechanisms of action of MIAs, which is critical for their rational use clinically.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA.
authors.forename: J M
authors.initials: JM
authors.lastname: Eichhorn
authors.forename: N
authors.initials: N
authors.lastname: Sakurikar
authors.forename: S E
authors.initials: SE
authors.lastname: Alford
authors.forename: R
authors.initials: R
authors.lastname: Chu
authors.forename: T C
authors.initials: TC
authors.lastname: Chambers
mesh_terms: D017209:Apoptosis; D016203:CDC2 Protein Kinase / Q000378:metabolism; D053148:Caspase 3 / Q000378:metabolism; D064199:Cdc20 Proteins / Q000378:metabolism; D004789:Enzyme Activation; D055785:Gene Knockdown Techniques; D006367:HeLa Cells; D006801:Humans; D008938:Mitosis; D050505:Mutant Proteins / Q000378:metabolism; D064549:Myeloid Cell Leukemia Sequence 1 Protein / Q000378:metabolism*; D010766:Phosphorylation; D059748:Proteolysis; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D051020:bcl-X Protein / Q000378:metabolism*
chemical_list: D064199:Cdc20 Proteins; C579095:MCL1 protein, human; D050505:Mutant Proteins; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D019253:Proto-Oncogene Proteins c-bcl-2; D011993:Recombinant Fusion Proteins; D051020:bcl-X Protein; C577488:CDC20 protein, human; D016203:CDC2 Protein Kinase; D053148:Caspase 3